Identification of PCR products – agarose gel electrophoresis

Adenine Thymine Guanine Cytosine

6. Identification of PCR products – agarose gel electrophoresis

Students who study this chapter will acquire the following specified learning outcomes:

Knowledge

The students understand the basics of gel electrophoresis

The students know the role of the solid support in electrophoresis.

The students list the requirements towards the solid support.

The students understand the sample preparation steps for the agarose gel electrophoresis.

The students list the factors influencing the migration of the DNA in the gel.

Skills

They students select appropriate molecular weight markers.

The students identify DNA fragments visualized in the solid support by molecular weight marker.

The students decide about the properties of the agarose gel for optimal performance in DNA fragment separation.

The students evaluate the results of the agarose gel electrophoresis experiment.

The students cast agarose gel.

Attitude

The students take effort to design a successful agarose gel electrophoresis experiment.

The students take care of adjusting the proper conditions for the agarose gel electrophoresis experiment.

The students document the gel electrophoresis experiment and accurately process the gel photo obtained.

Responsibility and autonomy

The students design their agarose gel electrophoresis experiments independently, taking into account the various factors that influence the mobility of the DNA fragments.

The students make effort to explore the further possibilities of the agarose gel electrophoresis in DNA investigations.

To verify the success of PCR, the products are visualized by means of electrophoresis as it was already demonstrated in Fig. 26. This procedure is based on the different mobility of ions in solution upon applying electric field. In aqueous solution itself, the difference in the migration ability of various ions is negligible and in addition, the increase of the temperature increases the flow of the solvent. Therefore, a solid matrix is applied to separate the ions. The 3D network of the pores within the solid support decreases the diffusion compared to the liquid and also enhances the separation efficiency.

However, the solid supports must fulfill several requirements to be applicable in electrophoretic systems:

(i) They should be hydrophilic, as the electrophoresis is carried out in aqueous buffer solution.

(ii) Chemical inertness is necessary. The solid support should not react with the substances applied for separation.

(iii) The solid support shall be neutral i.e. with no charges, which could interfere with the migration of the ions.

(iv) Adjustable pore size is an advantageous property of the solid support, as it provides flexibility to carry out the separation of ions with various properties.

(v) The solid support shall be physically rigid, it has e.g. to endure the transport form the electrophoresis tank to the documentation chamber.

(vi) It has also to be transparent to guarantee the visibility of the various dyes applied in electrophoresis.

(vii) It should not absorb pigments which are applied in electrophoresis.

The most abundant solid supports are gels made from agarose or polyacrylamide. As the former is applied more often for the investigation of DNA, the polyacrylamide gels will be discussed in the chapter dealing with proteins later. Agarose is a polysaccharide, purified from agar or agar-bearing marine algae. It is a linear polymer of the repeating unit of agarobiose. The building block (monomer) of the agarose is depicted in Fig. 32.

Figure 32. Agarobiose (4‐O‐β‐D‐galactopyranosyl‐3,6‐anhydro‐L‐galactose) is the monomeric unit of the agarose consisting of D-galactose and 3,6-anhydro-L-galactose units

It is a white solid powder, to be weighted on a balance for gel preparation.

It is then transferred into a buffer or water and heated up usually in a microwave oven until the solution becomes clear and homogeneous. In certain laboratories the buffer used in gel electrophoresis is added to this solution when it cools down to ~ 50°C in its 50 × concentrated form, in a way that the final concentration of the buffer should be the required one according to the protocol. Commonly TAE

of TBE buffers are used in agarose gel electrophoresis. The composition of the concentrated buffers is shown in Table 2.

As an easy exercise, calculate the final molar concentration of the constituents (for molecular weights and densities refer to external sources). Also check the price of 500 mL 50 × concentrated buffers, described in the table, based on the information available on the web in their ready form, and put together by the researcher.

Table 2. The composition of the concentrated buffers used for agarose gel preparation.

50 × concentrated TAE 5 × concentrated TBE 121 g of Tris base 54 g of Tris base 28.5 mL of acetic acid 27.5 g of boric acid 50 mL of 0.5 M EDTA 20 mL of 0.5 M EDTA

water up to 500 mL water up to 1 L

After mixing of the solution, the gel casting follows. The solution is transferred into a casting tank. A comb is placed in the cast to create wells for sample loading, as shown in Fig. 33. The liquid is then allowed to cool down below the gelling temperature. This temperature is dependent on the source of the agarose, usually it is in the 35–42 °C range. The three-dimensional matrix of the agarose gel is formed of the agarose polymers in supercoiled bundles. The gel is usually completely set within ~ 30 minutes.

Figure 33. Agarose gel casting. The comb has to be set properly to create the wells for sample loading. (Take care not to punch out the gel with the comb when it is set and when it is removed.) The scalper is used to cut the gel into pieces required for the gel electrophoretic experiment.

Pores and channels are formed in the three dimensional structure of the gel formed in this process, through which charged biomolecules can migrate. For investigation of DNA fragments, the most frequently applied technique is the agarose gel electrophoresis (AGE). The negatively charged DNA-molecules migrate towards the positive electrode. They are separated by their size in agarose gel: the shorter DNA fragments (lower molecular mass) migrate more quickly, while the longer ones more slowly. Naturally, the size of the pores and channels will determine the migration ability of the analytes. During the design of the agarose gel electrophoresis experiment both the pore size of the gel and the expected size of the DNA molecules to be separated shall be taken into account.

The larger is the DNA molecule the more effectively the matrix impedes its migrations. However, too small pores and channels will prevent the large DNA

molecules to be separated from each other, as they all migrate very slowly. In the contrary, the gel with an increased pore size will not be suitable for the separation of small size DNA fragments, as they all will migrate with the same maximal velocity. As a general guideline, the advised concentrations of agarose gels for the separation of DNA molecules of certain size ranges are provided in Table 3.

Table 3. The suggested concentration of agarose for separation of various DNA mixtures in AGE.

Agarose concentration (g / 100 mL) Optimal DNA resolution (kbp)

0.5 1 – 30

0.7 0.8 – 12

1.0 0.5 – 10

1.2 0.4 – 7

1.5 0.2 – 3

As an exercise, make a decision about the concentration of the gel for ideal separation of the PCR products shown in Fig. 26 (note that the DNA marker is needed for the identification of the size of the fragments – see later).

The set agarose gel is transferred into the horizontal electrophoresis tank, and it is immersed into the buffer solution with 1 × concentration. The level of the buffer solution shall be high enough to fully cover the gel. Make sure that the gel is placed in the right direction, i.e., the wells shall be close to the negative electrode, as the DNA molecules will migrate toward the positive electrode. In the next step the samples are loaded into the wells of the gel using digital

Finnpipettes for liquid handling (a wide range of such pipettes are on the market), as it is shown in Fig. 34.

Figure 34. Sample loading in for agarose gel electrophoresis. The volume of the sample is usually few L. The handling of such small volumes is done by precision digital Finnpipettes. Plastic tips are attached to the pipettes, which have to be replaced for each new sample. By means of this, the contamination of the original and the loaded samples can be avoided. (Notice the blue color of the DNA sample.)

DNA is well soluble in aqueous buffer solutions. This makes it difficult to load into the wells and prevent its immediate diffusion into the electrophoresis tank. To avoid this the density of the DNA solution has to be increased. For this purpose, glycerol or concentrated sucrose solution is used. Such materials are usually used in the "loading dye" solutions optimized for DNA sample loading

and visualization. The latter is important not only for safe sample loading but also to visually follow the migration of the DNA during AGE. Thus, various dyes (Fig. 35.) are also mixed into the loading dye solution, such as Bromophenol Blue, Xylene Cyanol, Orange G, etc.

Figure 35. The most common dyes applied in AGE for the visualization of DNA loading and migration.

The DNA sample solution is mixed with the 6 × or 10 × concentrated loading dye solutions prior to loading it into the wells of the agarose gel. As it can immediately be noticed from Fig. 35, all the applied dyes are negatively charged under the conditions of the AGE, thus, they will migrate in the same direction as the DNA. The different dyes migrate with a different velocity depending on the gel concentration. Nevertheless, apart from examining the behavior of the dyes in the specific setup, general experience may help conducting an AGE experiment.

According to the observations, Table 4. shows the relationship between the migration of the DNA and the dyes.

Bromophenol Blue Xylene Cyanol Orange G

Table 4. Observations on the relationship of the migration of DNA and pigments commonly applied in loading dyes during AGE.

Dye 0.5–1.5 g / 100 mL

agarose gel

2.0–3.0 g / 100 mL agarose gel

Xylene Cyanol 4–10 kbp 0.2–0.8 kbp

Bromophenol Blue 0.4–0.5 kbp < 0.15 kbp

Orange G < 0.1 kbp n.d.

When all the samples are properly loaded, the gel electrophoresis is initiated by applying a high voltage between the anode and cathode. For a common experiment, a 7 V / cm potential gradient factor is advised, which has to be multiplied by the distance between the two electrodes (in cm) for the proper potential. The start of the electrophoresis is accompanied by visible evolution of gas bubbles at the electrodes, as the electrolysis of the water occurs as a side-reaction. The electrophoresis can usually be terminated after 20–30 minutes, but the process also can be visually followed through the migration of the dye additives. A running AGE experiment is shown in Fig. 36. Because of the applied high voltage, it is very dangerous to touch the gel during the process. To prevent this, many electrophoretic instruments are already designed in a way that the electric circuit is disconnected by removing the cover from the electrophoresis tank – for safe operation.

As an exercise, try to identify the dyes on the gel and decide, whehter the experiment has to be stopped now if you would like to separate DNA fragments

in 3.0 – 6.0 kDa size range, supposed that 1 g / 100 mL concentration agarose gel (often denoted as 1 % in the literature) was used here.

Figure 36. The agarose gel in the course of AGE in a simple instrument. The red arrow indicates the direction of the migration of the DNA and the negatively charged dyes, while the actual position of the two applied dyes in the loading dye are shown by the blue and yellow arrows.

A care has to be taken of the precise conditions of the AGE experiment, since the buffer in the tank is warming up during the procedure. As the structure of the gel is held together with hydrogen bonds, it can be disrupted by warming up to its melting temperature close to 85–95 °C. In this case, the experiment will be unsuccessful. But warming up to ~50 °C will also soften the gel, so that it will be difficult to transfer it to the documentation phase, as well as the migration of the DNA is also altered in warm buffer. If necessary, the buffer has to be cooled.

After finishing the electrophoresis the DNA molecules have to be visualized in the gel. This is most often achieved by reacting the DNA with an intercalating reagent, ethidium bromide. Because of this property of ethidium ion, it is carcinogenic – thus, wearing gloves when working with it is a must!!! There are newly developed dyes for detecting DNA, such as GelRedTM, which is claimed to be safe to use, without carcinogenic effects. The ethidium ion becomes fluorescent, emitting orange light with a wavelength maximum of 605 nm when intercalated into the DNA double strand and irradiated with UV light (Fig. 37.).

The fluorescence of the dye itself is negligible in comparison to the intercalated one, but it causes a slight orange background.

A B

Figure 37. A) The structural formula of the ethidium bromide DNA intercalating agent. B) An agarose gel with DNA visualized by ethidium bromide.

The reagent can be supplied by immersing the ready gel into an ethidium-bromide solution, but it also can be added directly to the agarose in the same step of the preparation of the gel, as it was applied for the 50 × buffer. Ethidium bromide may be decomposed at high temperatures, therefore, it should be only

added when the agarose solution cools down to ~50 °C and then the solution has to mixed well to achieve homogeneous distribution of the dye. This latter method has several disadvantages. The main is the carcinogenicity of the reagent, which requires special handling of all the AGE reagents and instruments with gloves. It also has to mentioned that the ethidium ion is positively charged, thus it migrates in the opposite direction in the gel as compared to the DNA. This is well recognized in the gel shown in Fig. 37B. The background fluorescence in the lower part of the gel has disappeared, as there is no ethidium bromide. This phenomenon makes it more difficult to identify the small size (< 200 bp) DNA fragments if they run close to the bottom of the gel. In addition, the small size DNA can bind less fluorescent molecules, which further decreases its fluorescence. The ethidium ion intercalated to the DNA may also influence the migration behavior of the DNA fragment. On the other hand, staining the gel by immersing it into ethidium bromide solution for ~ 30 min is not as efficient as the direct involvement of the dye in the gel, and also it promotes slight diffusion of the DNA in the gel resulting in the decrease of the picture definition. The optimal solution has to be chosen for the relevant experiment. The result of every experiment has to be documented. For this purpose, in AGE several advanced gel documentation instruments have been developed. The one used in the laboratory of the author is depicted in Fig. 38. This instrument consists of a UV transilluminator and a digital camera system. The photos can be transferred to computers for further processing.

A B

Figure 38. A) An AGE gel documentation system B) An agarose gel documented, which needs always to be processed and labelled properly, e.g. as it is shown in Fig. 26.

The multiple bands usually in the first lane of the gel arise from a DNA mixture of known size DNA fragments. This is called DNA marker (or DNA ladder), based on which the size of the investigated DNA sample is estimated.

Running this marker only in a parallel with the sample in the same gel, the size of the unknown DNA fragment can be obtained properly, since the migration of the DNA molecules in the gel depend on various factors as mentioned above. Several DNA markers are available on market containing different ranges of sizes of the DNA fragments, as shown in Fig. 39.

Figure 39. Various markers from various suppliers can be chosen for the AGE experiments. Select always the appropriate one for identification of the investigated DNA.

It is worth mentioning that the migration of the DNA molecules in the agarose gel also depends on the shape of the DNA. Most of the markers consist of only linear DNA molecules. Thus, the bands of the marker aro only comparable with the bands of linear DNA. This is appropriate for detection of the PCR fragments. However, it was already mentioned, that plasmids are circular DNA molecules recognized by bacteria. These circular molecules can exist in several condensed supercoiled forms, topological isomers. Topoisomerase enzymes are able to interconvert between such topological states. A plasmid preparation usually contains superhelical (or supercoiled) form of the DNA, but depending on the skills of the researcher, and the protocol applied, the so called open circular (relaxed) form of the plasmid DNA also appears (Fig. 40.). The amount of the latter form can also be increased by introducing a single strand cleavage (nick) in the superhelical DNA.

GeneRuler^TM

100 bp DNA ladder GeneRuler^TM 1000 bp DNA ladder

A B

Figure 40. A) Different forms of bacterial plasmid DNA which can be converted into each other by the help or various enzymes. B) The result of the AGE experiment carried out with a plasmid DNA sample.

The circular DNA can also be linearized by introducing a double strand cleavage. Looking at Fig. 40. it can be easily suggested that the most compact superhelical form migrates most quickly in the agarose gel, while the bulky open circular form is usually very slow. The band of the linear form is usually detected between the two above forms.

DNA ladder pUC19 NColE7 0 min NColE7 15 min NColE7 30 min NColE7 45 min NColE7 60 min pUC19 GGNG 0 min GGNG 15 min GGNG 30 min GGNG 45 min GGNG 60 min pUC19 DNA ladder pUC19 KGNK 0 min KGNK 20 min KGNK 60 min KGNK 100 min KGNK 140 min pUC19 KGNG 0 min KGNG 20 min KGNG 60 min KGNG 100 min KGNG 140 min pUC19 GGNK 0 min GGNK 20 min GGNK 60 min GGNK 100 min GGNK 140 min

szuperhelikális

Monitoring questions

- Which method is suitable for easy verification of the success of PCR?

- How can be the various ions distinguished by an electrophoresis experiment?

- What is the most commonly used solid support in the electrophoretic separation of DNA mixtures?

- What is the basic principle of the separation of DNA molecules by agarose gel electrophoresis?

- What kind of agarose gel shall we prepare for the separation of a mixture containing large, and what kind for the separation of a DNA mixture containing small DNA molecules?

- What is the composition of the "loading dye"? What is the role of the pigments in the "loading dye"?

- How can the DNA molecules be detected in the agarose gel? Which important precautions have to be considered during this procedure?

- How can the DNA fragments be identified in the gel picture?

- Which properties of the DNA influence its migration in the agarose gel?

In document The biological tools of modern chemistry (Pldal 82-99)